Tumor targeting using anti-her2 immunoliposomes

We have generated anti-HER2 (ErbB2) immunoliposomes ILs), consisting of lon g circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragm ents, to provide targeted drug delivery to HER2-overexpressing cells. Immun oliposomes were constructed using a modular strategy in which components we re optimized for internalization and intracellular drug delivery. Parameter s included choice of antibody construct, antibody density, antibody conjuga tion procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitr o as determined by fluorescence microscopy, electron microscopy, and quanti tative analysis of fluorescent probe delivery. Delivery via ILs in HER2-ove rexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs s howed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no i ncrease in clearance, further confirming that ILs retain the long circulati on and non-immunogenicity of sterically stabilized liposomes. In five diffe rent HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxoru bicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significan tly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox + MAb or liposomal do x + MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes (P values from <0.0001 to 0.04 in eight separat e experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs an d non-targeted liposomal dox produced equivalent antitumor effects. Detaile d studies of tumor localization indicated a novel mechanism of drug deliver y for anti-HER2 ILs. Immunotargeting did not increase tumor tissue levels o f ILs vs. liposomes, as both achieved very high tumor localization (7.0-8.5 % of injected dose/g tissue) in xenograft tumors. However, histologic studi es using colloidal-gold labeled ILs demonstrated efficient intracellular de livery in tumor cells, while non-targeted liposomes accumulated within stro ma, either extracellularly or within macrophages. In the MCF7 xenograft mod el lacking HER2-overexpression, no difference in tumor cell uptake was seen , with both ILs and non-targeted liposomes accumulating within stroma. Thus , anti-HER2 ILs, but not non-targeted liposomes, achieve intracellular drug delivery via receptor-mediated endocytosis, and this mechanism is associat ed with superior antitumor activity. Based on these results, anti-HER2 immu noliposomes have been developed toward clinical trials. Reenginecring of co nstruct design for clinical use has been achieved, including: new anti-HER2 scFv F5 generated by screening of a phage antibody library for internalizi ng anti-HER2 phage antibodies; modifications of the scFv expression constru ct to support large scale production and clinical use; and development of m ethods for large-scale conjugation of antibody fragments with liposomes. We developed a scalable two-step protocol for linkage of scFv to preformed an d drug-loaded liposomes. Our final, optimized anti-HER2 ILs-dox construct c onsists of F5 conjugated to derivatized PEG-PE linker and incorporated into commercially available liposomal doxorubicin (Doxil (R)). Finally, further studies of the mechanism of action of anti-HER2 ILs-dox su ggest that this strategy may provide optimal delivery of anthracycline-base d chemotherapy to HER2-overexpressing cancer cells in the clinic, while cir cumventing the cardiotoxicity associated with trastuzumab + anthracycline. We conclude that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery, and that this strategy may also be appli cable to other receptor targets and/or using other delivered agents. (C) 20 01 Elsevier Science BY All rights reserved.